Gender dysphoria in twins: a register-based population study

Both genetic and environmental influences have been proposed to contribute to the variance of gender identity and development of gender dysphoria (GD), but the magnitude of the effect of each component remains unclear. We aimed to examine the prevalence of GD among twins and non-twin siblings of individuals with GD, using data derived from a large register-based population in Sweden over the period 2001–2016. Register data was collected from the Statistics Sweden and the National Board of Health and Welfare. The outcome of interest was defined as at least four diagnoses of GD or at least one diagnosis followed by gender-affirming treatment. A total of 2592 full siblings to GD cases were registered, of which 67 were twins; age at first GD diagnosis for the probands ranged from 11.2 to 64.2 years. No same-sex twins that both presented with GD were identified during the study period. The proportion of different-sex twins both presenting with GD (37%) was higher than that in same-sex twins (0%, Fisher’s exact test p-value < 0.001) and in non-twin sibling pairs (0.16%). The present findings suggest that familial factors, mainly confined to shared environmental influences during the intrauterine period, seem to contribute to the development of GD.

people with gender incongruence do not develop GD defined as a term in clinical settings. The few twin studies that have examined outcomes related to GD have shown a possible genetic influence, but with varying heritability or concordance rates. Coolidge et al. estimated heritability at 62% 10 , while Sasaki et al. found a heritability of 41% for adolescent assigned females (aF) and 11% for adult aF, and no genetic effects for assigned males (aM) 14 . Heylens et al. reported a concordance of 39.1% in MZ twins and 0% in DZ pairs 12 . Diamond found that the concordance among same-sex DZ pairs was 33% for aM and 23% for aF 11 .
In addition, these studies have some methodological limitations, such as small sample size 12,14 , heterogeneous population 11 and parent rating rather than self-rating 10 .
Beyond twin studies, a study has also reported familial cases of GD in 5 non-twin sibling or father-child pairs 16 . Likewise, a Spanish study on 995 consecutive transsexual probands reported 12 pairs of GD in non-twin siblings 17 . This study further showed that the probability that a sibling of a transsexual will also be transsexual was 4.48 times higher for siblings of male-to-female than for siblings of female-to-male transsexual probands, and 3.88 times higher for the brothers than for the sisters of transsexual probands.
Overall, evidence for a genetic etiology of GD remains precarious and is mainly compromised by the large heterogeneity in study design, outcome definitions, measures of assessing GD and the assessment of potential confounders across different studies. In the present study, we aimed to examine the prevalence of GD among same-and different-sex twins compared to a non-twin sibling cohort derived from a large register-based population in Sweden over the period 2001-2016.

Methods
Participants and procedure. Our source population consisted of all individuals who had received a GD diagnosis according to the 10th version of the International Classification of Diseases (ICD-10) between January 1, 2001, andDecember 31, 2016, and were at least 10 years old (N = 4374). Individuals who had also been diagnosed with the previous ICD-8 and ICD-9 GD diagnoses (N = 100) or had previously changed legal sex (N = 22) were excluded, given that these were not incident cases. Using the National Patient Register (NPR) and Prescribed Drugs Register (PDR), individuals with surgical or prescribed hormonal gender-affirming treatment prior to first diagnosis were also excluded (N = 166), in order to achieve higher validity of our definition, as some components of gender-affirming treatment could theoretically be given for indications other than gender dysphoria. The decision to have 2001 as the starting year of the follow-up period was partly because of the introduction of ICD-10 in Sweden in 1997 and partly because the outpatient NPR started in 2001. The study was designed in 2016 and that year was, therefore, the end of the follow-up period.
Individuals with GD were defined as those who received one GD diagnosis (F64.0 Transsexualism, F64.8 Other gender identity disorders, or F64.9 Gender identity disorder, unspecified) in the NPR on at least four occasions, or those who received at least one GD diagnosis followed by gender-affirming treatment. The first definition was utilized in order to include individuals with clinically significant GD who, for a variety of reasons, did not receive gender-affirming treatment, while the latter was used in order not to miss individuals with treated GD but with too short follow-up time to obtain four diagnoses. The definition, as proposed by Indremo et al. 18 , showed to be indicative of clinically relevant GD in the Swedish setting when compared with other register definitions.
We identified 2592 individuals with GD according to our definition, of which 1536 had at least one full sibling, which yielded 2601 siblings (Fig. 1). Twins were defined as full siblings born in the same year and month as the proband. Both twin and non-twin pairs are not unique given that a pair of two siblings (e.g., A and B) would appear twice, if they both have a GD diagnosis, because each individual would be proband for its sibling (A-B and B-A).
Register data was collected from the Statistics Sweden (SCB) and the National Board of Health and Welfare. All registers in Sweden use the 10-digit National Registration Number, a unique personal identifier assigned to all Swedish residents to allow linkage between registers. When retrieving data from the registers, all personal identifiers are replaced with consecutive numbers to secure anonymity. From the National Board of Health and Welfare, we retrieved data from the NPR, comprising information on primary and secondary ICD diagnoses from visits to specialist outpatient care since 2001 and inpatient health care since 1964, including surgeries, admission, and discharge dates. The inpatient register has nation-wide coverage since 1987, while the outpatient register started with a coverage between 8 and 18% during its first three years and reached 93% coverage by 2015. Sociodemographic data on birth dates, assigned sex at birth, change of legal sex, country of birth, and education level were retrieved from the Swedish Total Populations Register and linked to the NPR and PDR, that includes data on all redeemed medication since July 1, 2005. Data available from the Swedish Multi-Generation register allow the linkage of individuals born in 1932 and later, to parents, siblings and offspring 19

Results
Over the period 2001-2016, 2592 full siblings were registered, of which 67 were twins; one set of triplets (one proband with two "co-twins") was also identified ( Fig. 2), raising the number of twin pairs to 67 for the 66 probands.
The 66 probands were born between August 1950 and September 2003. Age at first GD diagnosis for the probands ranged from 11.2 to 64.2 years ( Table 1). The non-twin sibling pairs consisted of 1520 unique probands and a total of 2534 full siblings.
Among the 67 twin siblings of the probands, there were 10 (14.9%) with GD according to our definition. There were 40 probands with a same-sex twin and 27 probands with a different-sex twin. All 10 twin siblings with GD were twins to a different-sex proband, i.e., among the twins to a proband of the same assigned sex, none had GD (p < 0.001; Table 2). There were 4 full siblings with GD (0.16%) among the 2534 non-twin siblings. The mean follow-up time from the first GD diagnosis until the end of study period was 5.4 years, ranging from 1 to 14.8 years for the twins with GD.

Discussion
Based on registry data from a large Swedish population-based cohort, we compared the prevalence of GD among twin and non-twin siblings to persons with our definition of GD during a period of 16 years (2001-2016). No same-sex twins (and thus no MZ) with GD were identified. The prevalence in different-sex twin pairs was much higher than the prevalence in same-sex twins, as well as than that in non-twin sibling pairs. The different-sex pairs can, by definition, be only DZ twins, whereas the same-sex twin pairs can be either MZ or DZ. Since there were no twin siblings with GD among the 40 same-sex twin pairs, we can conclude that there are no concordant MZ twins, irrespective of the total number of MZ twins. By contrast, among the different-sex (i.e., DZ) twins there were 10 (37%) with GD. Interestingly, there were only 4 full siblings with GD (0.16%) among the 2534 non-twin siblings, a striking difference in concordance compared to the different-sex twin pairs, considering that they are genetically similar. This difference cannot be explained by genetics or shared childhood environments, pointing to a possible effect of intrauterine exposures. www.nature.com/scientificreports/ Both genetic, epigenetic and environmental factors have been proposed to account for the variance in gender identity 8 . However, the true magnitude of the effect of each component has not been clarified so far. Table 3 summarizes the literature findings on gender identity-related outcomes in twin and non-twin siblings. Some previous twin studies have reported higher concordance in MZ than in DZ twins 11,12 , while some studies have shown moderate genetic influences in gender variation 7,9 , and others have shown that heritability accounts for 11%-70% of gender variance 10,14,15 . The large variability in study design, study period, outcome definitions and    Our study had a different outcome than most other studies that reported on outcomes related to gender identity, gender role or cross-gender behavior, i.e., traits and not clinical diagnoses. Indeed, the only study in the literature that included a clinical definition and a clinical setting for GD, similar to ours, was that by Heylens et al. 12 That study, which showed a higher concordance in MZ than in DZ twins, was a hybrid study including 20 previously published case reports and 31 twin pairs that had attended a gender clinic and had been clinically assessed for gender identity disorder (GID), the latter being more comparable setting to our setting. The conclusion of higher concordance in MZ (39.1%) was heavily driven by the case reports, which are subject to publication bias. The 31 twin pairs that had a clinically assessed GID consisted of 6 MZ and 25 DZ pairs and the concordance rate in MZ pairs was 16.7% (1 out of 6 pairs) and 0% in DZ pairs. Most of the twins (N = 25) in that study came from a total of 561 children evaluated between 1976 and 2011 at the Gender Identity Service at the Centre for Addiction and Mental Health in Toronto, while three twin pairs came from the Gender Clinic of Ghent University Hospital during the period 1985-2010, from a pool of about 450 attendees and three twin pairs came from the Child and Adolescent Gender Clinic of Ghent University Hospital 12 . Interestingly, there were no concordant twins in this sample, including the 18 same-sex twin pairs (4 MZ and 14 DZ). Table 2. Gender dysphoria (GD) in twin and non-twin siblings to probands of same or different assigned sex.   www.nature.com/scientificreports/ Moreover, 28 of the twin pairs the study by Heylens et al. were children, and 25 of those were younger than 12 at the diagnosis of GD. In our study, the mean age at diagnosis was 25 and 32 years for discordant and concordant twin pairs, respectively, which consists an interesting major difference between these studies. Even though the age at diagnosis is by no means the same with age of onset of GD, the vast majority of the twins included in the study by Heylens et al. had a clearly early onset of GD and a GD diagnosis in childhood, which was not the case in our study. Of note is that previous studies have stipulated that the effect of genetics on gender identity may be prone to changes during lifetime 14 . In particular, heritability may be a non-fixed parameter with an attenuated effect on gender identity throughout adolescence and adult life 14,23 .
Another study with a similar outcome was the study by Sasaki et al. 14 , that used self-assessment of GID with criteria based on DSM-IV. The primary outcomes of the study were prevalence and heritability of GID but the authors also reported on the concordance for GID among MZ, DZ, same-and different-sex twin pairs. That study showed a higher concordance among same-sex twin pairs (20.7%) than different-sex twin pairs (8.8%). Although the outcome was similar to ours, the study was population based and the authors recognize, among other potential limitations, the fact that their definition of GID does not necessarily reflect a clinical GID diagnosis.
Prenatal sex hormones have been considered to play a significant role in brain sexual differentiation through permanent organizing effects during a critical period of fetal brain organization, especially during the last trimester of the pregnancy 14,[24][25][26] . These organizing structures are activated by sex hormones during puberty. Several studies have shown that sexually dimorphic brain structures seem to be more congruent with gender identity than the sex assigned at birth, possibly as a result of the effect of sex hormones, creating, thus, a restricted potential for change 27,28 . Indeed, our results of higher prevalence of GD in different-sex twins than that in same-sex twins and non-twin siblings in the present study, if replicated, provide some evidence that intrauterine factors, such as sex hormones produced by the co-twin, may influence the development of GD.
Some previous twin studies have provided evidence for a strong genetic influence in shaping gender identity. Candidate gene association studies have investigated whether functional variants in genes may alter sex hormone signaling, resulting in atypical sexual differentiation of the developing brains of those who will later experience GD 29,30 .
However, most studies have not used a stringent definition of GD as used in the present cohort, which may explain the null effect of heritability found herein. Indeed, the potential effect of genetics, if any, may apply to the development of gender identity as a trait, but not necessarily to the clinical diagnosis of GD. Future studies need to clarify the intricate interaction between genetics and environmental influences on gender identity and GD 26 .

Strengths and limitations.
In the present study we used, for the first time, registry data from a large Swedish population-based cohort over a long period (2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016) to study the familial aggregation of GD in the clinical setting, using a validated definition of GD. Our study with 67 twin pairs is larger than the one by Heylens et al., which is the largest available study with similar design studying clinical GD and which included 31 twin pairs. However, both studies are small and subject to random error. Moreover, the small power of the present study did not allow further sub-analyses, e.g., by age, to be conducted. Another limitation of our study was the lack of information about zygosity. However, it would be safe to assume that among the 40 same-sex pairs, approximately half of them are MZ pairs 31 , as almost one third of all twins are MZ 32 . The distribution of twin pairs in our study, i.e., the ratio between same-sex (n = 40) and different-sex (n = 27) pairs, was 1.5:1, which is in line with that reported by the Swedish Twin Registry 31 . The observation time from the first GD diagnosis varied from 1 to 15 years and thus we cannot exclude the possibility that some siblings and co-twins with shorter follow-up time could receive a GD diagnosis after the end of this study period and thus be misclassified as discordant. Lastly, the main outcome of this study was a clinically significant GD diagnosis according to our definition; thus, the present findings cannot provide assumptions on the whole picture of gender identity.

Conclusions
The present register-based study aimed to examine the concordance for GD diagnosis utilizing a cohort of twin and non-twin sibling pairs in Sweden. We found no concordance among same-sex twins, as well as higher concordance among different-sex twin than non-twin sibling pairs. Beyond any potential limitations and biases, our findings, if replicated, suggest that familial factors, mainly confined to environmental influences during the intrauterine period, are more likely to explain the development of GD. Future studies are deemed necessary to shed light into the intricate interaction between genetic and environmental factors, as well as into the potentially relevant molecular pathways that underlie these associations, if causal.

Data availability
Our study includes data from Swedish health care registers, which cannot be shared due to confidentiality issues. Data are available from the National Board of Health and Welfare in Sweden (registerservice@socialstyrelsen.se) and Statistics Sweden (https:// www. scb. se/ om-scb/ konta kta-oss/ stati stiks ervice/ fraga-oss).